- Email: [email protected]
Wheat gluten and its glutenin component: Viscosity, diffusion and sedimentation studies
ARCHIVES
OF
Wheat
BIOCHEMISTRY
AND
Gluten
and
BIOPHYSICS
97,
Its Glutenin and
the Northern
Component:
Sedimentation
N. W. TAYLOR From
(1962)
399-405
Viscosity,
Diffusion
Studies J. E. CLUSKEY
AND
Regional
Research
Laboratory:
Received
December
19, 1961
Peoria,
Illinois
Gluten from Ponca wheat was fractionated by precipitation with aqueous buffers into glutenin and the soluble gliadin fraction. The glutenin in pH 3.1 buffers had a high intrinsic viscosity, which increased markedly with lowering of ionic strength. It had a broad distribution of sedimenting components of high molecular weight and was highly heterogeneous in diffusion. Glutenin thus approximates a randomly coiled polyelectrolyte with a broad distribution of molecular size. INTRODUCTION
Wheat gluten is a mixture of several procomponents in electrophoresis (1, 2). One of these, previously designated a-l, is partly insoluble in salt or neutral solutions (1). This insoluble material, thus prepared in purified form, forms an elastic mass on precipitation similar to a rather stiff gluten ball. It is insoluble in 70% ethanol and consequently is closely related to the glutenin described by Osborne (3j. In this paper the a-1 fractions are called glutenin. Two glutenin fractions were isolated and then characterized by the physical methods indicated; their properties were compared with those of the original gluten and of the gliadin fraction. tein
MATERIALS
AND
METHODS
Methods for preparation of gluten and for electrophoresis were as previously reported (1, 4). One isolation of Poncs wheat gluten was used in this on other study, except for minor observations isolates as indicated. Al lactate was a cosmetic grade washed once with a small amount of water. All solvents were made to the required molarity by weighing out either the salt or base and then adding enough of the appropriate acid to make the pH 3.1. Solutions
were dialyzed several days in the cold before measurements were made. The concentrations of protein in solution for diffusion and sedimentation measurements were about 0.4% The isolated gluten was fractionated by titrating a 0.3% solution in 0.017 M Al lactate buffer to pH 4.8 with 0.2 M NaOH. The precipitated glutenin fraction (1~ glutenin) was collected after the mixture was left overnight in the cold. The two fractions were dialyzed against 0.01 M acetic acid, and then were lyophilized. Some of the 1X glutenin was reprecipitated from 0.3% solution three successive times (4~ glutenin) and was taken up in 0.017 M Al lactate buffer, pH 3.1, after each precipitation. Sedimentation rates were determined in a Spinco’ ultracentrifuge. Viscosities were determined in Cannon-Fenske No. 100 viscometers at 25.0” constant to O.Ol”C. Outflow times were recorded for several concentrations of protein and for the dialyzing solvent, These kinematic viscosities were not corrected for densities of the solutions. Concentrations of protein were determined by refractometry either in a Brice and Halwer differential refractometer (5) or from the Rayleigh interference pattern in a Spinco model H electrophoresis apparatus. To determine the specific refractive increment, wheat gluten was dialyzed severa1 days in a solvent, after which time known ‘Mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.
1 This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. 399
400
TAYLOR
AND CLUSKEY
FIG. 1. Electrophoretic patterns (ascending limb) of glut.en and its fractions. A and B in 0.017 M Al lactate buffer, pH 3.1; C and D in 0.03 M Na chloroacetate buffer, pH 3.1.
weights of solution and solvent were lyophilized in a vacuum flask and then dried to constant weight in the flask, at 95” in high vacuum. The weight of dry material from the solution was corrected for the weight of salts found in an amount of solvent containing the same weight of water. Diffusion curves were determined in a Spinco model H apparatus from Rayleigh fringe patterns. The fringes were measured with a Gaertner two-dimensional comparator reading to 0.002 mm. Boundaries were sharpened by withdrawing fluid there with a steel needle. The zero time was determined for several fringes by extrapolating (XJ,~ - X,)‘/Z’ to zero (6), when plotted against time, t. RESULTS SPECIFIC REFRACTIVE INCREMENT
The specific refractive increment of Ponca gluten was determined in pH 3.2 Na chloroacetate buffers of 0.003, 0.01, and 0.03 M salt. Results were 1.89, 1.88, and 1.87, respectively, in units of 100 ml./g. X 1O-3 at 1°C. in the Spinco model H instrument. At room temperature results were 1.88 and 1.89 in 0.003 and 0.01 M salt, respectively, in the Brice-Halwer instrument. The average value, 1.88 X 10e3 (100 ml./g.), was used to determine concentrations in all buffers with both instruments. FRACTIONATION
The glutenin fraction (1 x glutenin) recovered from one precipitation contained 34% of the original weight of gluten, and the gliadin fraction (IX gliadin) contained 56%.
Further purification of the glutenin fraction to obtain 4~ glutenin yielded 13% of the weight of original gluten as glutenin. Electrophoretic patterns of the gluten and fractions (Fig. 1) show that the IX glutenin is enriched in (X component; about 85% is present compared to 62% for gluten. The 4~ glutenin has about 95% of a-1 component, as observed in Na chloroacetate buffer, although it is difficult to judge where the tails of the a-1 component stop in the electrophoresis diagram. Some C(component remained in the 1X gliadin fraction. The electrophoretic patterns in Na chloroacetate buffer indicate that the glutenin fractions are mostly the a-1 component observed in this solvent (1). INTRINSIC VISCOSITIES
Intrinsic viscosities, [q], of Ponca gluten and its fractions were determined in Na lactate buffers at two ionic strengths (Table I). Ionic strength is greater than the moTABLE INTRINSIC
I
VISCOSITIES~ OF GLUTEN ITS FRACTIONS 1X fractions
Molarity of Ni;fi;y Gluten pH 3.i
Ghtenin
Gliadin
AND
4X fractions Mathem$2-a’ thesis
Ponca
Wichita
0.56 0.26
0.62
2.23 0.70
Glutenin
~__-___
0.003 0.03
0.57 0.26
1.14 0.43
a Units of intrinsic
0.21 0.16
viscosity are 100 ml./g.
WHEAT
larity of salt by 0.001 because of the contribution of ionized acid. The [v] of gluten was about twice as large in the lower ionic strength solvent. This effect is attributable to the glutenin component; its fractions showed a much greater increase of viscosity with lowering of ionic strength than did the gliadin fraction. Table I also compares a 4~ precipitated glut,enin from Wichita flour. This purified glutenin gave an increase in [v] that was relatively greater t,han for the IX glutenin. The [v] of the gluten of Wichita flour in 0.003 M Na lactate buffer was 0.65, only slightly greater than that for Ponca. [v] values were reproducible within about 5%. The variation with ionic strength was reversible for 1 x glutenin. In principle, intrinsic viscosities of mixtures of solutes are additive in proport,ion to weight fractions, and Table I shows the mathematical synthesis on this basis of [v] for gluten from the 1X fractions. Good agreement with the experimental values for gluten shows that no changes occurred in t’he proteins during the experiments. The intrinsic viscosities of gluten were also examined in other buffers. In Na chloroacetate buffers of pH 3.1 and 0.003,0.01, and 0.03 M salt, t,he [v] values were 0.59, 0.35, and 0.17, respectively. Precision here was only within about 15%. When the lactate and chloroacetate buffers are compared, it appears that the ionic strength is the major A
401
(3LUTEN
factor influencing [VI, while the specific anions are of minor importance. Ponca gluten was also examined in 0.017 M Al lactate buffer, a system used in electrophoresis. A different isolate from Ponca flour was used. The intrinsic viscosity of this isolate averaged 0.36 in Al lactate, as compared to 0.28 in 0.03 M Na lactate and 0.71 in 0.003 M Na lact’ate. The intermediate value of intrinsic viscosity in Al lact.ate buffer reflects a lower ionic strength than 0.1, its nominal value (1). SEDIMENTATION
BEHAVIOR
The sedimentation diagrams (7) of gluten and its fractions, showing the apparent proportion, g(s), of material sedimenting with a rate, s, were compared after 1 hr. at 47,660 r.p.m. (Fig. 2A). These curves have been normalized in such a manner that the areas for the fractions are proportional to their weight per cent in the gluten. The curves are neither corrected for concentration dependence nor for broadening by diffusion. Concentration dependence was virtually absent, as demonstrated by sedimentation runs with different concentrations of gluten (data not given) both in 0.03 M Na lactate and in 0.017 M Al lactate buffers. The diagrams for both buffers were identical. In other experiments broadening by diffusion was large for the slower sedimenting components. B
Sedimentation
FIG. 2. Sedimentation
diagrams of gluten and its fractions in 0.03 M Na lactate buffer after 64 min. at 47,660 r.p.m. In A, gluten and the 1X gliadin and 1X glutenin fractions are normalized to be proportional in area to their proportion in gluten. In B, the 4~ glutenin fraction is not normalized with respect to A.
402
TAYLOR
AND CLUSKEY
The IX glutenin (Fig. 2A) contains a faster sedimenting component near 6 s and has a broad distribution of sedimenting components. The 4~ glutenin (Fig. 2B) had a maximum at 6.0 s and was a very broad dispersion extending to high sedimentation rates. Of the total concentration of material, 88% was included below 58 s, and from earlier photographs it appeared that some even faster sedimenting material was present. In both the IX and 4~ glutenins there was evidence of a peak at about 2 s. This slow peak was largely eliminated in the 4X glutenin and was indicated to represent contaminants in the glutenin fractions. The mathematical synthesis (not shown) of the sedimentation diagram of gluten from
the diagrams of the two fractions agrees fairly well with the gluten curve in the region less than 5 s, but not in the region of faster sedimentation. The glutenin had lost the plateau region by this stage and concentration had built up toward the bottom of the cell. For this reason the glutenin was difficult to compare quantitatively with the slower sedimenting gluten and IX gliadin fraction. DIFFUSION
The diffusion coefficients of gluten and its fractions were determined in several solvents. The experimental points on each curve (Fig. 3) are values of (XJjz Xj)2/224t = D in Gosting’s terminology
A
I I I I 0.4 0.1 0.2 0.3 Fraction of Concentration
I I I I I 0 0.1 0.2 0.3 0.4 to Boundary Center ()/-j/J)
(
FIG. 3. Diffusion curves of gluten and its fractions. D is explained in the text. A, in 0.03 M Na lactate; B; in 0.003 M Na lactate buffer. The solvent (0) and solution (0) sides of the diffusing boundaries are given separately.
403
WHEBT GLUTEK (6). This function would be the diffusion coefficient in the ideal case with no concentration dependence, heterogeneity, or flow interaction. The values of D are the experimental ones determined from the distance between the fringe, j, and the midconcentration point, J/2. A comparison of results in 0.03 M Na lactate (Fig. 3A) and 0.003 M Na lactate (Fig. 3B) shows that in the latter solvent there was a large concentration dependence, as indicated by the failure of the two sides of the boundary to give identical results. Further, when concentrations of all three proteins were reduced to one-fourth, the diffusion curves approached closer to the results in 0.03 M Na lactate. Two of these curves are shown by the dotted lines in Fig. 3B. Apparently the variance from results in 0.03 M Na lactate is almost entirely due to concent’ration dependence of diffusion. In 0.03 M Na lactate where concentration dependence is negligible, the diffusion coefficient of 1 x glutenin is lower than that of gluten, that of 4~ glutenin is still lower and that for 1X gliadin is higher. The mathemat’ical synthesis of DA (D at the boundary center as described below) from the two IX fractions, 1.01 x lo-’ sq. cm./ sec., agrees well with the experimental value for gluten, 0.97 X 10U7. The diffusion curve of gluten in 0.017 M Al lactate buffer (data not given) was experimentally identical to that in 0.03 M Na lactate. There was only a small concentration dependence in the latt’er solvent also. In various Na chloroacetate buffers the diffusion curves of gluten were similar to results in Na lactate buffers of the corresponding molarity, except that the curves were slightly lower (data not given j. In the 0.03 M Na chloroacetate buffer, the curve for the solvent side was somewhat higher than the curve for the solution side. From these experiments and from the intrinsic viscosities, it was concluded that the 0.03 M Na chloroacetate buffer was a very poor solvent for gluten. The heterogeneity of the diffusing materials can be estimated by comparing their diffusion coefficients calculated by different
TABLE II DIFFUSION
COEFFICIEKTS GLUTEN AND ITS
OF PONCA FRACTIONS
Material
DA x 107, SO.. cm./sec. at 1°C.
Gluten 1X gliadin 1X glutenin 4X glutenin
0.97 2.1 0.53 0.30
WHEAT
D.&f/Da
2.0 1.2 1.9 1.9
methods. The values obtained by ext,rapolating the diffusion curves in Fig. 3 to zero abscissa are equivalent to DA , the diffusion coefficient determined by the reduced height-area met.hod, according to Creeth (8). He extrapolated 2 dDt as a function of z”, where Z is the normalized distance from boundary center, because this relationship should be linear at low 2. Results on his plot were experimentally equivalent to the extrapolated values in Figs. 3A and 3B, within the accuracy desired here. The reduced second moment diffusion coefficients, D, , were calculated by Svensson’s method (9j for the four preparations shown in Fig. 3, and the ratios IL),/ D-4 then were obtained (Table II). Deviation of this ratio from one is a measure of het,erogeneity of the diffusing material. The gluten and glutenin fractions are highly heterogeneous in diffusion. The lx gliadin fraction is less heterogeneous than the gluten or glutenin fractions, although in electrophoresis it is the more heterogeneous fraction. DISCUSSIOTU’ FRACTIONATIOS
OF GLUTEN
These studies are concerned with the acidextracted purified gluten protein from nearly completely defatted wheat flour. The purified material seems representative of native gluten in that it will form a gluten ball and is identical in electrophoresis to material which bakes good bread (10). The measured properties probably only approximate those of the entire native material. There is some 15% of crude gluten protein lost in the extraction, which may be significant to the physical properties of the gluten.
404
TAYLOR AND CLUSKEY
Fractionation of the isolated gluten does not affect the proteins appreciably. Good agreement of the physical properties of the gluten with the mathematical syntheses of the properties of the fractions establishes this point. The glutenin fractions are continuously variable with successive purifications. The 4x glutenin has a lower diffusion coefficient and a higher intrinsic viscosity compared with the 1 x glutenin than can reasonably be ascribed to removal of the other electrophoretic components. Such fractionation is expected in view of the observed heterogeneity of sedimentation of the glutenin fractions, and also of the calculated heterogeneity of diffusion noted below. Apparently there is a glutenin component in gluten having an electrophoretic mobility of a-1, a broad distribution of sedimenting species, and a broad distribution of species with respect to intrinsic viscosity and diffusion. Our glutenin fractions are, in this respect, isolates of part of this component and are, to some extent, contaminated with other components of gluten.
When this evidence is considered, the conclusion that glutenin molecules or particles are expanded in low ionic strength solution seems firmly established. The expansion of the glutenin molecules indicates the presence of an insufficient secondary structure to hold a constant shape. Glutenin may be a freely flexible, randomly coiled polyelectrolyte, or it may, like bovine serum albumin, have some secondary structure but insufficient to maintain a rigid shape in the acid medium (13). While there is not much evidence to differentiate these possibilities, a random coil character is favored by the elasticity shown by wet glutenin when insoluble and in a neutral pH medium. Udy (14) noted that the viscosity of gluten solutions in dilute acetic acid varied with dilution similar to a polyelectrolyte. Our results confirm his idea, and they demonstrate the glutenin component to have this property. By comparison, our gliadin fractions have a low, relatively constant, intrinsic viscosity that perhaps indicates they are comparatively globular proteins.
POLYELECTROLYTE NATURE OF GLUTENIN
MOLECULAR WEIGHTS AND HETEROGENEITY
The intrinsic viscosities of the glutenin fractions were greater in solvents of lower ionic strength. Such behavior can be attributed to expansion of the molecules or particles provided other effects can be ruled out. In low ionic strength solvents, electroviscous effects may contribute to solution viscosities, but the contributions to intrinsic viscosities are relatively minor in theory (11) and cause negligible changes in the intrinsic viscosity of a globular protein, ribonuclease (12). Neither aggregation nor disaggregation is an important factor here, because there is evidence that molecular weight distribution does not change. Both the sedimentation diagrams and diffusion curves of gluten were experimentally identical in the two solvents, 0.017 M Al lactate and 0.03 M Na lactate, and indicate a constant molecular weight distribution. The diffusion curves of both gluten and its fractions appeared nearly identical also in 0.03 and 0.003 M Na lactate buffers, when protein concentration was sufficiently low.
Both gliadin and gluten are heterogeneous in such physical properties as solubility (15, 16)) sedimentation (17, 18)) and diffusion (17, 19). Our results confirm these findings for sedimentation and diffusion, and they further demonstrate that glutenin, also, is heterogeneous in these properties. The glutenin component is not completely precipitable by our conditions. Not only does the gliadin fraction contain considerable material with an electrophoretic mobility of Q, but successive precipitations result in successively less material having smaller diffusion coefficients and higher intrinsic viscosities in a given solvent. Because part of the glutenin is apparently left in the gliadin fraction and because the least soluble glutenin is likely not even isolated in our preparations, the fractions studied here do not show the full range of heterogeneity of glutenin. Thus, quantitative estimates of heterogeneity in glutenin are not possible as yet. The molecular weight of the 4x-precipi-
WHEAT
tated glutenin calculated from the peak of the sedimentation curve and the diffusion coefficient, D, , is 1 million. Evidently very much higher and lower molecular weight materials are also present. Similarly for gluten, the molecular weight determined is 100,000; for the gliadin fraction, 60,000. Jones et al. (20) have determined the weight-average molecular weights of glutenin and two components of gliadin using the approach-to-sedimentation-equilibrium technique. Their weight-average molecular weight for 4~ glutenin was several millions. Our value of 1 million, which is a different kind of average, is not inconsistent with theirs. The question arises whether glutenin is a physical aggregate. As its molecular weight is unchanged by several strong protein solvents (20), it appears not to be an aggregate but rather a cystine cross-linked polymer (21). In summary, the available evidence indicates that glutenin is a protein which is highly heterogeneous in particle size, with a range up to a high molecular weight. It behaves as a flexible, randomly coiled polyelectrolyte. It is the major contributor to the rheological properties of gluten in solution because of its high viscosity, and it is probably the major factor in the viscoelastic properties of wet gluten. REFERENCES 1. JONES, R. W., TAYI.OR, N. W., .4ND SENTI, F. R., Arch. Biochem. Biophys. 84,363 (1959). 2. WOYCHIK, J. H., BOUXQY, J. A., AND DIMLER, I-
GLUTEN
405
R. J., Arch. Biochem. Biophys. 94, 477 (1961). 3. OSBORSE, T. B., “The Vegetable Proteins,” 2nd ed. Longmans, Green and Co., London, 1924. 4. CLUSKEY, J. E., TAYLOR, X. W., CHARLEY, H., .~XD SENTI, F. R., Cereal Chem. 38, 325 (1961). 5. BRICE, B. 8. AND HALWER, M., J. Opt. sot. Am. 41, 1033 ( 1951). 6. GOSTINC, L. J., Adunncrs in Protein Chem. 11, 429 (1956). 7. WILLIAMS, J. W., V.4v HOLDE, K. E., BALDWIN, R. I,., .~KD FCJIT.4, H., Chem. Re~x. 58, 715 (1958). 8. CREETH, J. M., J. Phys. Chem. 62,66 (1958). 9. SVENSSOK, H., Attn. Chem. Scnntl. 5, 1410, (1951). 10. LUSEYA, C. V., Cereal Chem. 27, 167 (1950). 11. BOOTH, F., Proc. Roy. Sot. (London), A203, 533 ( 1950). 12. BUZZELL, J. G. ASD TASFORI), C., J. Phys. Chcm. 60, 1204 f 1956). 13. YASC, J. T., ASD FOSTER, J. F., J. Am. Chem. Sot-. 76, 1588 (1954). 14. ITor, 13. C., Cereal Chem. 30, 288 (1953). 15. HAUGAARD, G., .~ND JOHXSO~-, A. H., Corn@. rend. traL>. lab. f’nrlsberg 18(2), 1 (1930). 16. 1~&ALLA, 4. G., AND ROSE, R. C., Can. J. Rc~.senrch12, 346 (1935). 17. M&ALLA, A. G., APED GRALEX, S.. Can. .I. Research 20, 130 (1942). 18. KREJCI, I,. E., ASD SYEDBERG, T., J. Bm. Chcm. Ser. 57, 946 (1935). 19. 1,.4mf, o., AND Po~.sos. A., Biochem. J. 30, 528 (1936). 20. Jor~s, R. R., BABCOCK, G. E., TAYLOR, N. W., ASD SESTI, F. R., Arch. Biochem. Biophys. 94,483 (1961). 21. NIELSEN, H. c., BABCOCK. CT. E., .4h‘D SEYTI, P. R., Arch. Biochem. Bi(jphys. 96, 252 (1962).
OF
Wheat
BIOCHEMISTRY
AND
Gluten
and
BIOPHYSICS
97,
Its Glutenin and
the Northern
Component:
Sedimentation
N. W. TAYLOR From
(1962)
399-405
Viscosity,
Diffusion
Studies J. E. CLUSKEY
AND
Regional
Research
Laboratory:
Received
December
19, 1961
Peoria,
Illinois
Gluten from Ponca wheat was fractionated by precipitation with aqueous buffers into glutenin and the soluble gliadin fraction. The glutenin in pH 3.1 buffers had a high intrinsic viscosity, which increased markedly with lowering of ionic strength. It had a broad distribution of sedimenting components of high molecular weight and was highly heterogeneous in diffusion. Glutenin thus approximates a randomly coiled polyelectrolyte with a broad distribution of molecular size. INTRODUCTION
Wheat gluten is a mixture of several procomponents in electrophoresis (1, 2). One of these, previously designated a-l, is partly insoluble in salt or neutral solutions (1). This insoluble material, thus prepared in purified form, forms an elastic mass on precipitation similar to a rather stiff gluten ball. It is insoluble in 70% ethanol and consequently is closely related to the glutenin described by Osborne (3j. In this paper the a-1 fractions are called glutenin. Two glutenin fractions were isolated and then characterized by the physical methods indicated; their properties were compared with those of the original gluten and of the gliadin fraction. tein
MATERIALS
AND
METHODS
Methods for preparation of gluten and for electrophoresis were as previously reported (1, 4). One isolation of Poncs wheat gluten was used in this on other study, except for minor observations isolates as indicated. Al lactate was a cosmetic grade washed once with a small amount of water. All solvents were made to the required molarity by weighing out either the salt or base and then adding enough of the appropriate acid to make the pH 3.1. Solutions
were dialyzed several days in the cold before measurements were made. The concentrations of protein in solution for diffusion and sedimentation measurements were about 0.4% The isolated gluten was fractionated by titrating a 0.3% solution in 0.017 M Al lactate buffer to pH 4.8 with 0.2 M NaOH. The precipitated glutenin fraction (1~ glutenin) was collected after the mixture was left overnight in the cold. The two fractions were dialyzed against 0.01 M acetic acid, and then were lyophilized. Some of the 1X glutenin was reprecipitated from 0.3% solution three successive times (4~ glutenin) and was taken up in 0.017 M Al lactate buffer, pH 3.1, after each precipitation. Sedimentation rates were determined in a Spinco’ ultracentrifuge. Viscosities were determined in Cannon-Fenske No. 100 viscometers at 25.0” constant to O.Ol”C. Outflow times were recorded for several concentrations of protein and for the dialyzing solvent, These kinematic viscosities were not corrected for densities of the solutions. Concentrations of protein were determined by refractometry either in a Brice and Halwer differential refractometer (5) or from the Rayleigh interference pattern in a Spinco model H electrophoresis apparatus. To determine the specific refractive increment, wheat gluten was dialyzed severa1 days in a solvent, after which time known ‘Mention of firm names or trade products does not imply that they are endorsed or recommended by the Department of Agriculture over other firms or similar products not mentioned.
1 This is a laboratory of the Northern Utilization Research and Development Division, Agricultural Research Service, U. S. Department of Agriculture. 399
400
TAYLOR
AND CLUSKEY
FIG. 1. Electrophoretic patterns (ascending limb) of glut.en and its fractions. A and B in 0.017 M Al lactate buffer, pH 3.1; C and D in 0.03 M Na chloroacetate buffer, pH 3.1.
weights of solution and solvent were lyophilized in a vacuum flask and then dried to constant weight in the flask, at 95” in high vacuum. The weight of dry material from the solution was corrected for the weight of salts found in an amount of solvent containing the same weight of water. Diffusion curves were determined in a Spinco model H apparatus from Rayleigh fringe patterns. The fringes were measured with a Gaertner two-dimensional comparator reading to 0.002 mm. Boundaries were sharpened by withdrawing fluid there with a steel needle. The zero time was determined for several fringes by extrapolating (XJ,~ - X,)‘/Z’ to zero (6), when plotted against time, t. RESULTS SPECIFIC REFRACTIVE INCREMENT
The specific refractive increment of Ponca gluten was determined in pH 3.2 Na chloroacetate buffers of 0.003, 0.01, and 0.03 M salt. Results were 1.89, 1.88, and 1.87, respectively, in units of 100 ml./g. X 1O-3 at 1°C. in the Spinco model H instrument. At room temperature results were 1.88 and 1.89 in 0.003 and 0.01 M salt, respectively, in the Brice-Halwer instrument. The average value, 1.88 X 10e3 (100 ml./g.), was used to determine concentrations in all buffers with both instruments. FRACTIONATION
The glutenin fraction (1 x glutenin) recovered from one precipitation contained 34% of the original weight of gluten, and the gliadin fraction (IX gliadin) contained 56%.
Further purification of the glutenin fraction to obtain 4~ glutenin yielded 13% of the weight of original gluten as glutenin. Electrophoretic patterns of the gluten and fractions (Fig. 1) show that the IX glutenin is enriched in (X component; about 85% is present compared to 62% for gluten. The 4~ glutenin has about 95% of a-1 component, as observed in Na chloroacetate buffer, although it is difficult to judge where the tails of the a-1 component stop in the electrophoresis diagram. Some C(component remained in the 1X gliadin fraction. The electrophoretic patterns in Na chloroacetate buffer indicate that the glutenin fractions are mostly the a-1 component observed in this solvent (1). INTRINSIC VISCOSITIES
Intrinsic viscosities, [q], of Ponca gluten and its fractions were determined in Na lactate buffers at two ionic strengths (Table I). Ionic strength is greater than the moTABLE INTRINSIC
I
VISCOSITIES~ OF GLUTEN ITS FRACTIONS 1X fractions
Molarity of Ni;fi;y Gluten pH 3.i
Ghtenin
Gliadin
AND
4X fractions Mathem$2-a’ thesis
Ponca
Wichita
0.56 0.26
0.62
2.23 0.70
Glutenin
~__-___
0.003 0.03
0.57 0.26
1.14 0.43
a Units of intrinsic
0.21 0.16
viscosity are 100 ml./g.
WHEAT
larity of salt by 0.001 because of the contribution of ionized acid. The [v] of gluten was about twice as large in the lower ionic strength solvent. This effect is attributable to the glutenin component; its fractions showed a much greater increase of viscosity with lowering of ionic strength than did the gliadin fraction. Table I also compares a 4~ precipitated glut,enin from Wichita flour. This purified glutenin gave an increase in [v] that was relatively greater t,han for the IX glutenin. The [v] of the gluten of Wichita flour in 0.003 M Na lactate buffer was 0.65, only slightly greater than that for Ponca. [v] values were reproducible within about 5%. The variation with ionic strength was reversible for 1 x glutenin. In principle, intrinsic viscosities of mixtures of solutes are additive in proport,ion to weight fractions, and Table I shows the mathematical synthesis on this basis of [v] for gluten from the 1X fractions. Good agreement with the experimental values for gluten shows that no changes occurred in t’he proteins during the experiments. The intrinsic viscosities of gluten were also examined in other buffers. In Na chloroacetate buffers of pH 3.1 and 0.003,0.01, and 0.03 M salt, t,he [v] values were 0.59, 0.35, and 0.17, respectively. Precision here was only within about 15%. When the lactate and chloroacetate buffers are compared, it appears that the ionic strength is the major A
401
(3LUTEN
factor influencing [VI, while the specific anions are of minor importance. Ponca gluten was also examined in 0.017 M Al lactate buffer, a system used in electrophoresis. A different isolate from Ponca flour was used. The intrinsic viscosity of this isolate averaged 0.36 in Al lactate, as compared to 0.28 in 0.03 M Na lactate and 0.71 in 0.003 M Na lact’ate. The intermediate value of intrinsic viscosity in Al lact.ate buffer reflects a lower ionic strength than 0.1, its nominal value (1). SEDIMENTATION
BEHAVIOR
The sedimentation diagrams (7) of gluten and its fractions, showing the apparent proportion, g(s), of material sedimenting with a rate, s, were compared after 1 hr. at 47,660 r.p.m. (Fig. 2A). These curves have been normalized in such a manner that the areas for the fractions are proportional to their weight per cent in the gluten. The curves are neither corrected for concentration dependence nor for broadening by diffusion. Concentration dependence was virtually absent, as demonstrated by sedimentation runs with different concentrations of gluten (data not given) both in 0.03 M Na lactate and in 0.017 M Al lactate buffers. The diagrams for both buffers were identical. In other experiments broadening by diffusion was large for the slower sedimenting components. B
Sedimentation
FIG. 2. Sedimentation
diagrams of gluten and its fractions in 0.03 M Na lactate buffer after 64 min. at 47,660 r.p.m. In A, gluten and the 1X gliadin and 1X glutenin fractions are normalized to be proportional in area to their proportion in gluten. In B, the 4~ glutenin fraction is not normalized with respect to A.
402
TAYLOR
AND CLUSKEY
The IX glutenin (Fig. 2A) contains a faster sedimenting component near 6 s and has a broad distribution of sedimenting components. The 4~ glutenin (Fig. 2B) had a maximum at 6.0 s and was a very broad dispersion extending to high sedimentation rates. Of the total concentration of material, 88% was included below 58 s, and from earlier photographs it appeared that some even faster sedimenting material was present. In both the IX and 4~ glutenins there was evidence of a peak at about 2 s. This slow peak was largely eliminated in the 4X glutenin and was indicated to represent contaminants in the glutenin fractions. The mathematical synthesis (not shown) of the sedimentation diagram of gluten from
the diagrams of the two fractions agrees fairly well with the gluten curve in the region less than 5 s, but not in the region of faster sedimentation. The glutenin had lost the plateau region by this stage and concentration had built up toward the bottom of the cell. For this reason the glutenin was difficult to compare quantitatively with the slower sedimenting gluten and IX gliadin fraction. DIFFUSION
The diffusion coefficients of gluten and its fractions were determined in several solvents. The experimental points on each curve (Fig. 3) are values of (XJjz Xj)2/224t = D in Gosting’s terminology
A
I I I I 0.4 0.1 0.2 0.3 Fraction of Concentration
I I I I I 0 0.1 0.2 0.3 0.4 to Boundary Center ()/-j/J)
(
FIG. 3. Diffusion curves of gluten and its fractions. D is explained in the text. A, in 0.03 M Na lactate; B; in 0.003 M Na lactate buffer. The solvent (0) and solution (0) sides of the diffusing boundaries are given separately.
403
WHEBT GLUTEK (6). This function would be the diffusion coefficient in the ideal case with no concentration dependence, heterogeneity, or flow interaction. The values of D are the experimental ones determined from the distance between the fringe, j, and the midconcentration point, J/2. A comparison of results in 0.03 M Na lactate (Fig. 3A) and 0.003 M Na lactate (Fig. 3B) shows that in the latter solvent there was a large concentration dependence, as indicated by the failure of the two sides of the boundary to give identical results. Further, when concentrations of all three proteins were reduced to one-fourth, the diffusion curves approached closer to the results in 0.03 M Na lactate. Two of these curves are shown by the dotted lines in Fig. 3B. Apparently the variance from results in 0.03 M Na lactate is almost entirely due to concent’ration dependence of diffusion. In 0.03 M Na lactate where concentration dependence is negligible, the diffusion coefficient of 1 x glutenin is lower than that of gluten, that of 4~ glutenin is still lower and that for 1X gliadin is higher. The mathemat’ical synthesis of DA (D at the boundary center as described below) from the two IX fractions, 1.01 x lo-’ sq. cm./ sec., agrees well with the experimental value for gluten, 0.97 X 10U7. The diffusion curve of gluten in 0.017 M Al lactate buffer (data not given) was experimentally identical to that in 0.03 M Na lactate. There was only a small concentration dependence in the latt’er solvent also. In various Na chloroacetate buffers the diffusion curves of gluten were similar to results in Na lactate buffers of the corresponding molarity, except that the curves were slightly lower (data not given j. In the 0.03 M Na chloroacetate buffer, the curve for the solvent side was somewhat higher than the curve for the solution side. From these experiments and from the intrinsic viscosities, it was concluded that the 0.03 M Na chloroacetate buffer was a very poor solvent for gluten. The heterogeneity of the diffusing materials can be estimated by comparing their diffusion coefficients calculated by different
TABLE II DIFFUSION
COEFFICIEKTS GLUTEN AND ITS
OF PONCA FRACTIONS
Material
DA x 107, SO.. cm./sec. at 1°C.
Gluten 1X gliadin 1X glutenin 4X glutenin
0.97 2.1 0.53 0.30
WHEAT
D.&f/Da
2.0 1.2 1.9 1.9
methods. The values obtained by ext,rapolating the diffusion curves in Fig. 3 to zero abscissa are equivalent to DA , the diffusion coefficient determined by the reduced height-area met.hod, according to Creeth (8). He extrapolated 2 dDt as a function of z”, where Z is the normalized distance from boundary center, because this relationship should be linear at low 2. Results on his plot were experimentally equivalent to the extrapolated values in Figs. 3A and 3B, within the accuracy desired here. The reduced second moment diffusion coefficients, D, , were calculated by Svensson’s method (9j for the four preparations shown in Fig. 3, and the ratios IL),/ D-4 then were obtained (Table II). Deviation of this ratio from one is a measure of het,erogeneity of the diffusing material. The gluten and glutenin fractions are highly heterogeneous in diffusion. The lx gliadin fraction is less heterogeneous than the gluten or glutenin fractions, although in electrophoresis it is the more heterogeneous fraction. DISCUSSIOTU’ FRACTIONATIOS
OF GLUTEN
These studies are concerned with the acidextracted purified gluten protein from nearly completely defatted wheat flour. The purified material seems representative of native gluten in that it will form a gluten ball and is identical in electrophoresis to material which bakes good bread (10). The measured properties probably only approximate those of the entire native material. There is some 15% of crude gluten protein lost in the extraction, which may be significant to the physical properties of the gluten.
404
TAYLOR AND CLUSKEY
Fractionation of the isolated gluten does not affect the proteins appreciably. Good agreement of the physical properties of the gluten with the mathematical syntheses of the properties of the fractions establishes this point. The glutenin fractions are continuously variable with successive purifications. The 4x glutenin has a lower diffusion coefficient and a higher intrinsic viscosity compared with the 1 x glutenin than can reasonably be ascribed to removal of the other electrophoretic components. Such fractionation is expected in view of the observed heterogeneity of sedimentation of the glutenin fractions, and also of the calculated heterogeneity of diffusion noted below. Apparently there is a glutenin component in gluten having an electrophoretic mobility of a-1, a broad distribution of sedimenting species, and a broad distribution of species with respect to intrinsic viscosity and diffusion. Our glutenin fractions are, in this respect, isolates of part of this component and are, to some extent, contaminated with other components of gluten.
When this evidence is considered, the conclusion that glutenin molecules or particles are expanded in low ionic strength solution seems firmly established. The expansion of the glutenin molecules indicates the presence of an insufficient secondary structure to hold a constant shape. Glutenin may be a freely flexible, randomly coiled polyelectrolyte, or it may, like bovine serum albumin, have some secondary structure but insufficient to maintain a rigid shape in the acid medium (13). While there is not much evidence to differentiate these possibilities, a random coil character is favored by the elasticity shown by wet glutenin when insoluble and in a neutral pH medium. Udy (14) noted that the viscosity of gluten solutions in dilute acetic acid varied with dilution similar to a polyelectrolyte. Our results confirm his idea, and they demonstrate the glutenin component to have this property. By comparison, our gliadin fractions have a low, relatively constant, intrinsic viscosity that perhaps indicates they are comparatively globular proteins.
POLYELECTROLYTE NATURE OF GLUTENIN
MOLECULAR WEIGHTS AND HETEROGENEITY
The intrinsic viscosities of the glutenin fractions were greater in solvents of lower ionic strength. Such behavior can be attributed to expansion of the molecules or particles provided other effects can be ruled out. In low ionic strength solvents, electroviscous effects may contribute to solution viscosities, but the contributions to intrinsic viscosities are relatively minor in theory (11) and cause negligible changes in the intrinsic viscosity of a globular protein, ribonuclease (12). Neither aggregation nor disaggregation is an important factor here, because there is evidence that molecular weight distribution does not change. Both the sedimentation diagrams and diffusion curves of gluten were experimentally identical in the two solvents, 0.017 M Al lactate and 0.03 M Na lactate, and indicate a constant molecular weight distribution. The diffusion curves of both gluten and its fractions appeared nearly identical also in 0.03 and 0.003 M Na lactate buffers, when protein concentration was sufficiently low.
Both gliadin and gluten are heterogeneous in such physical properties as solubility (15, 16)) sedimentation (17, 18)) and diffusion (17, 19). Our results confirm these findings for sedimentation and diffusion, and they further demonstrate that glutenin, also, is heterogeneous in these properties. The glutenin component is not completely precipitable by our conditions. Not only does the gliadin fraction contain considerable material with an electrophoretic mobility of Q, but successive precipitations result in successively less material having smaller diffusion coefficients and higher intrinsic viscosities in a given solvent. Because part of the glutenin is apparently left in the gliadin fraction and because the least soluble glutenin is likely not even isolated in our preparations, the fractions studied here do not show the full range of heterogeneity of glutenin. Thus, quantitative estimates of heterogeneity in glutenin are not possible as yet. The molecular weight of the 4x-precipi-
WHEAT
tated glutenin calculated from the peak of the sedimentation curve and the diffusion coefficient, D, , is 1 million. Evidently very much higher and lower molecular weight materials are also present. Similarly for gluten, the molecular weight determined is 100,000; for the gliadin fraction, 60,000. Jones et al. (20) have determined the weight-average molecular weights of glutenin and two components of gliadin using the approach-to-sedimentation-equilibrium technique. Their weight-average molecular weight for 4~ glutenin was several millions. Our value of 1 million, which is a different kind of average, is not inconsistent with theirs. The question arises whether glutenin is a physical aggregate. As its molecular weight is unchanged by several strong protein solvents (20), it appears not to be an aggregate but rather a cystine cross-linked polymer (21). In summary, the available evidence indicates that glutenin is a protein which is highly heterogeneous in particle size, with a range up to a high molecular weight. It behaves as a flexible, randomly coiled polyelectrolyte. It is the major contributor to the rheological properties of gluten in solution because of its high viscosity, and it is probably the major factor in the viscoelastic properties of wet gluten. REFERENCES 1. JONES, R. W., TAYI.OR, N. W., .4ND SENTI, F. R., Arch. Biochem. Biophys. 84,363 (1959). 2. WOYCHIK, J. H., BOUXQY, J. A., AND DIMLER, I-
GLUTEN
405
R. J., Arch. Biochem. Biophys. 94, 477 (1961). 3. OSBORSE, T. B., “The Vegetable Proteins,” 2nd ed. Longmans, Green and Co., London, 1924. 4. CLUSKEY, J. E., TAYLOR, X. W., CHARLEY, H., .~XD SENTI, F. R., Cereal Chem. 38, 325 (1961). 5. BRICE, B. 8. AND HALWER, M., J. Opt. sot. Am. 41, 1033 ( 1951). 6. GOSTINC, L. J., Adunncrs in Protein Chem. 11, 429 (1956). 7. WILLIAMS, J. W., V.4v HOLDE, K. E., BALDWIN, R. I,., .~KD FCJIT.4, H., Chem. Re~x. 58, 715 (1958). 8. CREETH, J. M., J. Phys. Chem. 62,66 (1958). 9. SVENSSOK, H., Attn. Chem. Scnntl. 5, 1410, (1951). 10. LUSEYA, C. V., Cereal Chem. 27, 167 (1950). 11. BOOTH, F., Proc. Roy. Sot. (London), A203, 533 ( 1950). 12. BUZZELL, J. G. ASD TASFORI), C., J. Phys. Chcm. 60, 1204 f 1956). 13. YASC, J. T., ASD FOSTER, J. F., J. Am. Chem. Sot-. 76, 1588 (1954). 14. ITor, 13. C., Cereal Chem. 30, 288 (1953). 15. HAUGAARD, G., .~ND JOHXSO~-, A. H., Corn@. rend. traL>. lab. f’nrlsberg 18(2), 1 (1930). 16. 1~&ALLA, 4. G., AND ROSE, R. C., Can. J. Rc~.senrch12, 346 (1935). 17. M&ALLA, A. G., APED GRALEX, S.. Can. .I. Research 20, 130 (1942). 18. KREJCI, I,. E., ASD SYEDBERG, T., J. Bm. Chcm. Ser. 57, 946 (1935). 19. 1,.4mf, o., AND Po~.sos. A., Biochem. J. 30, 528 (1936). 20. Jor~s, R. R., BABCOCK, G. E., TAYLOR, N. W., ASD SESTI, F. R., Arch. Biochem. Biophys. 94,483 (1961). 21. NIELSEN, H. c., BABCOCK. CT. E., .4h‘D SEYTI, P. R., Arch. Biochem. Bi(jphys. 96, 252 (1962).